This R21 application combines the expertise of two independent laboratories to address a pivotal question regarding our understanding of hematopoietic ontogeny- the developmental origin of hematopoietic stem cells (HSC). In the mouse embryo, primitive hematopoietic progenitors arise in the yolk sac during gastrulation (E7.0-E8.5). While definitive blood cells differentiate in the fetal liver beginning at E.9.5, HSC with the ability to engraft adult recipientsfirst arise within the embryo proper only at E10. Recently an "embryonic" HSC that provides long-term engraftment of newborn, but not adult, recipients has been identified at E9.0 in both yolk sac and embryo proper. It is not known if these HSC arise from intraembryonic (AGM) or extraembryonic (yolk sac) sites. In preliminary experiments, J. Palis has identified hematopoietic cells with high proliferative potential exclusively within the yolk sac of the pre-circulation mouse embryo (E8.25). We hypothesize that embryonic HSC also arise from extraembryonic sites during gastrulation. In the First Aim of this proposal, we will use an in utero transplantation model to determine the developmental origin of mammalian hematopoietic stem cells.Yolk sac and embryo proper cells from accurately staged mouse embryos between E7.5 - E9.5 will be transplanted directly into the livers of E14.5 W42 fetuses. This unique transplantation system will maximize our ability to identify HSC activity within the early post-implantation embryo. In the Second Aim of this proposal, we will study the temporal and spatial kinetics of engraftment of hematopoietic organs by embryonic and fetal hematopoietic stem cells. These experiments will take advantage of phenotypically defined E9.5 yolk sac and E14.5 liver donor stem cell populations expressing enhanced green fluorescent protein A better understanding of the initiation of mammalian hematopoiesis will provide insights into the ontogeny, regulation and expansion of HSC, as well as the origin of leukemias and bone marrow failure syndromes. This knowledge will ultimately lead to improvements in bone marrow transplantation for the curative treatment of congenital anemias, genetic diseases and many childhood and adult cancers.